Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L

Four tissues from the blue mussel, Mytilus edulis L., were examined for the presence of nuclear metallothionein (MT), and the nuclear:cytosolic (N:C) MT ratios and nuclear MT:DNA ratios investigated. Gill, digestive gland, gonad and posterior adductor muscle tissues were dissected, homogenized and subjected to differential centrifugation in order to isolate the nuclear and cytosolic fractions, which were then analyzed for MT and DNA. MT was present in all samples of the nuclear fractions from all four tissues. The nuclear MT concentration was either lower or the same as the cytosolic MT concentration from the same tissue. The mean N:C MT ratio of the digestive gland was significantly lower than that of the gill. The mean nuclear MT:DNA ratio of the digestive gland was significantly higher than that of the gill and posterior adductor muscle. In addition to being the first report of nuclear MT in bivalves, we showed that N:C MT ratios and nuclear MT:DNA ratios differ among tissues of the same organism. This raises important questions concerning the regulation of nuclear MT concentrations and the role of nuclear MT in metal regulation and DNA protection.     

Relevance of purine catabolism to hypoxia and recovery in euryoxic and stenoxic marine invertebrates,particularly bivalve molluscs

1. Activities of xanthine dehydrogenase (XDH) and xanthine oxidase (XOD) were measured in a variety of euryoxic and stenoxic marine molluscs.2. Euryoxic bivalves contain only XDH activity which, unlike the mammalian enzyme, is not converted to XOD during anoxic exposure.3. XOD activity was detected predominantly in stenoxic bivalves such as Pecten maximus, Placopeclen magellanicus, and in the cephalopod Loligo opalescens. Although extremely variable, XOD activity increased 4-fold in Cardium edule and 13-fold in Pecten maximus during anoxic exposures of 56 hr and 0.5 hr respectively.4. The data suggest that euryoxic species may tolerate anoxic-normoxic transitions in part by possessing a form of XDH that resists conversion to XOD (a source of Superoxide radicals responsible for ischemia-reperfusion tissue injury in mammals).5. XDH activities in Carcinus maenas digestive gland are sufficient to account fully for the urate reported to accumulate during hypoxia.     

青蛤形态学初步观察

青蛤是帘蛤科中分布较广的一种埋栖双壳贝类。壳近圆形,两侧近等,无小月面,有外韧带,主齿三枚,前闭壳肌痕呈半月形,后闭壳肌痕呈椭圆形。肌肉系统由闭壳肌、足伸缩肌、外套膜肌、水管肌、足肌等组成;消化系统分为消化道和消化腺两部分。消化盲囊呈绿色,为主要的消化腺体;鳃是青蛤主要呼吸器官,同时,外套膜也起到辅助呼吸的作用;循环系统为开管式,具后动脉球,血液中含有血清蛋白而使之成为无色液体;排泄系统由肾脏和围心腔腺组成;青蛤是雌雄异体,生殖期间,精巢为乳白至乳黄色,卵巢为粉红色;系统比较简单,具有三对神经节。     

Growth and reproduction of the bay scallop, Argopecten irradians (Lamarck) at its southern distributional limit

Growth and reproduction in a Florida population of bay scallop, Argopecten irradians (Lamarck), existing at the southern limit of its distribution, was monitored over a 3-yr period. Somatic growth accompanied increasing water temperature between May and August, when maximum mantle, digestive gland, and adductor muscle weights and indexes were observed. From July through September, reproductive development was correlated with a decline in adductor muscle weight and index. Maximum gonad weight, gonad index, and oocyte diameter were found in late September and early October. Spawning commenced at this time in conjunction with decreasing water temperature.In comparison to more northern populations, the reproductive cycle of the Florida bay scallop occurs later in the year and is characterized by a higher gametogenic initiation temperature, a decreased maximum gonad index and decreased oocyte diameter, and a shift in reproductive energy resources from digestive gland (available food) to adductor muscle (pre-stored reserves). Increased metabolic rate associated with higher temperature and coupled with decreased food supply results in the Florida bay scallop having less energy available for reproduction, ultimately limiting its southern distribution.     

Detection and characterization of gamete-specific molecules in Mytilus edulis using selective antibody production

The mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26-AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.     

Ultrastructure of the mushroom body: digestion during metamorphosis of Utterbackia imbecillis (Bivalvia: Unionidae)

Abstract. Larvae of the freshwater mussel Utterbackia imbecillis metamorphose to juveniles either during their attachment to a host fish, or in vitro in a culture medium. This transformation includes degeneration of larval structures and development of the juvenile morphology. Early in metamorphosis the cells comprising the larval mantle enlarge and project into the mantle cavity, forming a structure referred to as the mushroom body. Its cells, which are ultrastructurally very similar to digestive cells of adult bivalves, are involved in pinocytosis or phagocytosis of the larval adductor muscle and of tissue from the host fish that is enclosed between the larval shells. Ingested material is passed from pinosomes to heterophagosomes which in turn fuse with heterolysosomes, where final degradation of ingested material occurs. Acid phosphatase activity was detected in heterophagosomes and heterolysosomes of all animals examined. In larvae that metamorphosed in vitro , the apical cytoplasm of the cells of the mushroom body, and the extracellular spaces among them, also exhibited acid phosphatase activity. Larvae reared on a host fish accumulated substantial deposits of lipids and glycogen within larval mantle cells during metamorphosis, whereas larvae reared in vitro did not. The larval mantle cells which constitute the mushroom body appear to be the primary sites of intracellular digestion of the larval adductor muscle and host tissue during metamorphosis.     

THE ADDUCTOR AND RETRACTOR MUSCLES OF THE VELIGER OF PECTEN MAXIMUS (L.) (BIVALVIA)

In Pecten maximus (L.), retractor and adductor muscles becomefunctional in the early veliger larva. The twelve-day-old veligerhas four pairs of velar retractors, three pairs of retractorsattached to the posterior body wall and an anterior adductor.The pediveliger has in addition, pedal retractor muscles anda posterior adductor. The retractors consist of striated muscle:the adductors have both smooth and striated portions. The retractorsattach near the hinge, branch to a greater or lesser extent,then attach to specific areas of the velum, posterior body walland foot. Some features of the branching and of the dispositionof points of attachment form a pattern which exhibits mirrorsymmetry about the plane between the two shell valves. Thispattern is characteristic of the species. It is deduced thatretraction and protraction of the velum result from co-ordinatedsequences of muscle contractions. ^*Present address: Forest Products Research Centre, P.O. Box1358, Boroko, Papua New Guinea. (Received 15 June 1984;     

Storage metabolism in the Pacific oyster (Crassostrea gigas) in relation to summer mortalities and reproductive cycle (west coast of France)

We describe seasonal changes in the biochemical composition of digestive gland, adductor muscle and gonad and surrounding mantle area in Crassostrea gigas from the Western Atlantic coast of France. Seasonality in histology of storage tissues and glycogen storage capacity in isolated vesicular cells were also studied. Proteins, the main muscle components did not contribute to the gametogenetic effort. Glycogen and lipids were stored in the digestive gland, gonad and surrounding mantle area during the wintering period and the gonad and surrounding mantle area represented the main storage compartment supplying the reproductive effort. Gametogenesis in spring and summer was associated with an increase in lipid and protein contents and took place at the expense of glycogen reserves. Histological study of storage tissue in the gonad led us to define four seasonal stages of storage tissue development. In vitro, glycogen storage capacity in isolated vesicular cells was high from November to March and markedly reduced during gametogenesis, decreasing below detectable levels after spawning. This physiological state should be taken into account with relation to summer mortalities occurring in commercial growing areas.     

Assessment of the state of the Japanese scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) based on morphological and biochemical parameters

The results of an integrated examination of the state of the scallopMizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) are presented. In mollusks of different ages, shell height was measured; in animals of commercial size (over 100 mm), some size and weight characteristics (annual increment of shell and adductor muscle and soft tissue weight) were determined. The morphology of the digestive gland and gills was studied. In the adductor muscle and digestive gland, the concentration of heavy metals (HMs) (Hg, Cu, Zn, Mn, Pb, Cd, Ni, Co, and Cr) was determined. In the digestive gland, metallothionein and reduced glutathione concentration was also determined, as was the activity of glutathione-dependent enzymes (glutathione peroxidase and glutathione reductase). In scallops collected outside Alekseeva Bight, the linear growth rate and adductor muscle weight were on average 1.3 and 1.7 times greater, respectively, than in those collected in the bight. In scallop organs, numerous histomorphological alterations were revealed: digestive cell vacuolization and hemocyte infiltration of the digestive gland, hyperplasia and vacuolization of the respiratory epithelium, and connective tissue hypertrophy in gill filaments. The biochemical parameters of scallops from Alekseeva Bight differed substantially from those of mollusks collected outside the bight. We conclude that one of the factors negatively affecting the state of theM. yessoensis population in Alekseeva Bight is the contamination of the bight with HMs, especially mercury. This is consistent with the results of chemical analysis of bottom sediments and tissues of two mytilid species,Modiolus kurilensis andCrenomytilus grayanus, specimens of which were collected in the bight together with the scallops [3].     

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.     

Cellular cadmium distribution in the common winkle, Littorina littorea (L.) determined by X-ray microprobe analysis and histochemistry

Various tissues of common winkles, Littorina littorea (L.), experimentally exposed to cadmium (Cd) chloride were examined using light and electron microscopy and their elemental composition determined by X-ray microanalysis and histochemistry. Membrane granules in gill epithelial cells with paddle cilia contain carbonates, phosphates and sulphides associated with different cations in different types of granules. Traces of Cd have been found only in those granules containing sulphur and iron. Nephrocytes also contain small amounts of this metal in the cytoplasm of excretory cells. X-ray microanalysis reveals that concretions of basophilic cells are minor sites for Cd sequestration while BTAN-ASSG stain for unbound Cd indicates that most of the Cd is located within the lysosomes of digestive cells in association with proteins. Low amounts of the metal have been evidenced in the granules of epithelial mantle cells rich in sulphur. The results also indicate that hemocytes contain granules of calcium phosphate and iron sulphide. Cd is also associated to sulphur rather than to phosphate. These hemocytes may act as Cd carrier from gills to kidney and digestive gland. A hypothetical pathway for Cd accumulation and detoxification is suggested.     

Effects of the toxic dinoflagellate, Gymnodinium catenatum on hydrolytic and antioxidant enzymes, in tissues of the giant lions-paw scallop Nodipecten subnodosus

This study documents effects of the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison, on juvenile farmed (5.9+/-0.39 cm) giant lions-paw scallop Nodipecten subnodosus. Scallops were fed bloom concentrations of toxic dinoflagellate G. catenatum for 7 h. The effect of the toxic dinoflagellate in different tissues was determined by analysis of antioxidant enzymes (catalase, superoxide dismutase, gluthathione peroxidase), thiobarbituric acid reactive substances (lipid peroxidation), and hydrolytic enzymes (proteases, glycosidases, phosphatases, lipases, and esterases). Histopathological photos record the effects of the toxic dinoflagellate in various tissues. The results show that juvenile lions-paw scallops produce pseudo-feces, partially close their shell, increase melanization, and aggregate hemocytes. Several enzymes were affected and could serve as biological markers. In general, the adductor muscle was not affected. In the digestive gland, some enzymes could be the result of defensive and digestive processes. Gills and mantle tissue were markedly affected because these sites respond first to toxic dinoflagellates, leading to the idea that proteolytic cascades could be involved.     

Swimming performance, metabolic rates, and their correlates in the Iceland scallop Chlamys islandica

The dramatic escape response of some scallops is modified by reproductive investment and by acclimation temperature. Despite considerable knowledge of the physiology of the escape response, functional links between escape response performance, organismal rates of oxygen uptake, and tissue metabolic capacities are little known. We measured oxygen consumption rates (standard, maximal, and aerobic scope), escape behavior (initial and repeat performance), tissue mass, condition index, protein content, and tissue metabolic capacities in the Iceland scallop Chlamys islandica to examine links between these parameters. Postexercise oxygen consumption rates were positively linked to contraction rate (repeat test) and to pyruvate kinase activity in the adductor muscle but negatively linked to digestive gland wet mass. Swimming behavior was mainly related to activity of glycolytic enzymes, and enzymatic activities were related to anatomic parameters. Scallop behavior and physiology change with size, both within our samples and on a larger scale. Small scallops showed more intense swimming activity and had higher arginine kinase activities but lower glycolytic enzyme activities in their adductor muscle than larger scallops. This corresponds to the ontogenetic change in susceptibility to predation and in habitat use observed in C. islandica.     

Age-dependence of metabolism in mussels Mytilus edulis (L.) from the White Sea

Age structure, natural mortality and growth, as well as age- and size-dependent changes in parameters of energy metabolism were studied in blue mussels Mytilus edulis (L.) from the White Sea. Mussels were sampled in August (Summer sample, SS) and October (Autumn sample, AS) and contained animals of three size groups, 2-9 years old. Field data showed an increase of mortality of mussels and strong decrease in growth rates after 6 years of age. Absolute tissue growth increment (AI) reconstructed from winter growth marks on the shells decreased with age and was strongly size-dependent, while relative tissue growth increment (RI) did not depend on size of the animals. Respiration rates and citrate synthase activity demonstrated power regression versus tissue weight with regression coefficients -0.231 and -0.170, respectively. After weight correction both parameters showed a decrease with increasing age. ATP and phosphagen levels also showed a pronounced decrease in animals older than 5-6 years despite considerable differences in the absolute values of both parameters in SS and AS. pH(i) in mussels was also age-dependent and decreased with increasing age after 5 years. In air exposed mussels, pH(i) was reduced only at young age such that pH(i) was low and constant within the whole age range. Our data give evidence that aerobic metabolic rate in M. edulis from the studied population declines when animals reach an age of about 6 years. The decrease in oxygen consumption reflects the drop in mitochondrial respiration, which is mirrored by the decrease in CS activity. A concomitant fall in ATP turnover may include a downregulation of the mechanisms of acid-base regulation. pH(i) will then approach equilibrium indicated by lower pH(i) values in older animals. Our data suggest that intrapopulational comparisons of physiological parameters in mussels should take into account age composition of compared samples.     

The structure and distribution of ceramide aminoethylphosphonates in the oyster (Ostrea gigas).

1. Ceramide aminoethylphosphonate was isolated from the adductor, gills, mantle and viscera of oysters. 2. After drastic acid hydrolysis of the lipid, aminoethylphosphonic acid was the only water-soluble carbon-phosphorous compound detected. 3. The main fatty acids of ceramide aminoethylphosphonates were hexadecanoic acid (77-90%) and 2-hydroxy hexadecanoic acid (13-15%). 4. Hexadeca-4-sphingenine, octadeca-4-sphingenine and octadeca-4,8-sphingadienine were identified as the major long chain base components. However, the ratio of the three bases was characteristic for each tissue; the adductor muscle contains primarily hexadeca-4-sphingenine, and the viscera, octadeca-4,8-sphingadienine. The gills and mantle contain the three bases in approximately equal concentration. 5. The main molecular species in the adductor muscle was hexadecanoyl-hexadeca-4-sphingenyl 2-aminoethylphosphonate, while in the viscera hexade-canoyl-octadeca-4,8-sphingenyl 2-aminoethylphosphonate predominated.     

Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.